Single-cell analysis of the physiology of mechanosensation in bacteria

Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels.

Isolation and characteristics of a bacteriophage for bacillus stearothermophilus

A bacteriophage (TØ3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60°C, the optimum growth temperature of the host. At 60°C the phage has a latent period of 18 minutes and a burst size of about 200. The phage is comparatively thermostable in broth. The half life of the phage is 400 minutes at 60°C, 120 minutes at 65°C, 40 minutes at 70°C and 12 minutes at 75°C. The activation energy for the heat inactivation of TØ3 is 56,000 cal. The buoyant density of TØ3 in a cesium chloride density gradient is 1.526.

Electron micrographs of TØ3 indicate that the phage has a regular hexagonal shaped head 57 mμ long. The morphology of the head is compatible with icosahedral symmetry. Each edge of the head is 29 mμ long, and there are 6 or 7 subunits along each edge. The tail of TØ3 is 125 mμ long and 10 mμ wide. There are about 30 cross striations that are spaced at 3.9 mμ intervals along the tail.

The DNA of phage TØ3 has a melting temperature of 88.5°C. Heat denatured TØ3 DNA can be extensively annealed in a high ionic strength environment. The buoyant density of TØ3 DNA in a cesium chloride density gradient is 1.695. TØ3 DNA contains: 42.7% guanine plus cytosine, as determined from the melting temperature; 43% guanine plus cytosine, as determined from the buoyant density; and 40.2% guanine plus cytosine, as determined by chromatographic separation and spectrophotometric estimation of the bases. The molecular weight of TØ3 DNA is 16.7 X 10⁶ as determined from the band width of the TØ3 DNA concentration distribution in a cesium chloride density gradient. Electron microscopy of TØ3 DNA revealed a single linear molecule that is 11.7 μ long. This corresponds to a molecular weight of 22.5 X 10⁶.

Heat denatured TØ3 DNA forms two bands in a cesium chloride density gradient, one at a density of 1.707 and the other at a density of 1.715. After the separated bands are mixed and annealed in the centrifuge cell, the renatured TØ3 DNA forms a single band at a density of 1.699. These results indicate that the two complementary strands of TØ3 DNA have different buoyant densities in cesium chloride, presumably because they have different base compositions.

The characteristics of TØ3 are compared with those of other phages. A hypothesis is presented for a relationship between the base composition of one strand of TØ3 DNA and the amino acid composition of the proteins of TØ3.